This invention relates to a method for producing a recombinant viral expression vector. More particularly, this invention relates to a method for incorporating a selected gene coupled with a baculovirus polyhedrin promoter into a baculovirus genome to produce a recombinant baculovirus expression vector capable of expression of the selected gene in a host insect cell.
Recent advances in recombinant DNA technology have facilitated the isolation of specific genes or parts thereof and their transfer to bacteria, yeast or viruses. The transferred or modified gene(s) is replicated and propagated as the transformed microorganisms or viruses replicate and, as a result, the genetically altered recombinant may then have the capacity to produce the gene product for which the transferred gene sequences encode.
The transfer and expression of genes, or portions thereof, between viruses, eukaryotes and prokaryotes is possible because the DNA of all living organisms is chemically similar in that it is composed of the same four nucleotides. The basic differences reside in the sequence of the nucleotides in the genome of the organism. The nucleotide sequences are arranged in codons (nucleotide triplets) which code for specific amino acids with the coding relationship between amino acid sequence and nucleotide sequence being essentially the same for all organisms.
The DNA is organized into genes which are comprised of protein encoding genes (i.e., "structural genes") and control regions (usually referred to as the "promoter") that mediate initiation of expression of the structural gene. In general, the enzyme RNA polymerase is activated by the promoter such that as it travels along the structural gene, it transcribes encoded information into a messenger ribonucleic acid (mRNA). The mRNA contains recognition sequences, signals for ribosome binding, and signals for translational start and stop. Recent advances in the genetic analysis of the role of important transcriptional signals in the promoter regions of genes (which are usually described as the 5' flanking region of protein coding genes) have facilitated the ability to selectively remove or alter DNA sequences to study their function and role in expression, and to remove certain of these sequences to study their function in heterologous biological systems such as a host-vector system.
Eukaryotic promoters are usually characterized by two conserved sequences of nucleotides whose locations and structural similarity to prokaryotic promoter sequences (Breathnach & Chambon, Ann. Rev. Biochem. 50, 349-383 (1981)) suggest involvement in the promotion of transcription. The first is a sequence rich in the nucleic acids adenine and thymine (the Goldberg-Hogness, "TATA," or "ATA" box) which is located 20-30 base pairs upstream from the RNA initiation site (the cap site which is the transcriptional start site for the mRNA) and is characterized by a concensus sequence (5'-TATAA-ATA-3'). The second region is the CCAAT box (Efstratiadis, et al., Cell 21, 653-668 (1980)), which is located 70-90 base pairs upstream from the cap site of some genes and has the canonical sequence 5'-GG(C/T)CAATCT-3' (Benoist, et al., Nucleic Acids Res. 8, 127-142 (1980)). The development of techniques for removing and altering these sequences (Nathan and Smith, Ann. Rev. Biochem. 44, 273-293 (1975); Weber, et al.In : D. D. Brown and C. F. Fox (Eds.), Developmental Biology Using Purified Genes, ICN-UCLA Symposium on Molecular and Cellular Biology (New York, Academic Press, 1981) pp. 367-385) has made it possible to separate genes from their promoter regions or portions thereof in order to study their function in heterologous biological systems.
This has been accomplished by the use of restriction endonuclease enzymes and cloning to produce recombinant DNA molecules and the controlled removal or alteration of the cloned nucleotide sequences by in vitro or site-specific mutagenesis. Restriction endonucleases are hydrolytic enzymes capable of catalyzing the site-specific cleavage of DNA molecules. The site of restriction enzyme activity is determined by the presence of a specific nucleotide sequence and is termed the recognition site for a given restriction endonuclease. Many restriction enzymes have been isolated and classified according to their recognition site. Some restriction endonucleases hydrolyze the phospho-diester bonds on both DNA strands at the same point to produce blunt ends, while others hydrolyze bonds which are separated by a few nucleotides from each other to produce free single-stranded regions at the end of each DNA molecule. These single-stranded ends are self-complementary and may be used to rejoin the hydrolyzed DNA or another or heterologous DNA sequences with the same complementary single-stranded sequences.
Restriction sites are relatively rare, however the general use of restriction endonucleases has been greatly improved by the availability of chemically synthesized double-stranded oligonucleotides containing the desired restriction site sequence. Virtually any naturally occurring, cloned, genetically altered or chemically synthesized segment of DNA can be coupled to any other segment by attaching an oligonucleotide containing the appropriate recognition sites to the ends of the DNA molecule. Subjecting this product to the hydrolytic action of the appropriate restriction endonuclease produces the requisite complementary ends for coupling the DNA molecules.
Recognition sites for specific restriction enzymes are usually randomly distributed, therefore, cleavage by a restriction enzyme may occur between adjacent codons, within a codon or at some random site in the gene. While there are many possible variations on this scheme, it is important to note that techniques are available for inserting DNA sequences in the proper location and orientation with respect to a promoter region to allow expression of those sequences.
Potentially, any DNA sequence can be cloned by inserting a foreign DNA sequence into a cloning vehicle or vector molecule to construct an artificial recombinant molecule or composite sometimes called a chimera or hybrid. For most purposes, the cloning vehicle utilized is a duplex extrachromosomal DNA sequence comprising an intact replicon such that the recombinant molecule can be replicated when placed into bacteria or yeast by transformation. Cloning vehicles commonly in use are derived from viruses and plasmids associated with bacteria and yeast.
Recent advances in biochemistry and recombinant DNA technology have led to the construction of cloning vehicles or vectors containing "heterologous" DNA. The term "heterologous" refers to DNA that codes for polypeptides ordinarily not produced by the cell susceptible to transformation but which are incorporated into the genome of the cell(s) when the recombinant is introduced into the cell(s) by transformation. The transformant is thereafter isolated and cultured, thus obtaining large populations of the cell(s) which include copies of the foreign gene which may or may not be expressed in that particular cell. These populations provide a renewable source of the recombinant for further manipulations, modifications and transfer to other vectors.
Once the gene or desirable portions thereof have been cloned and or biochemically modified in the desired manner or other biochemically modified or genomic genes have been inserted in such a manner as to facilitate their expression, they are then transferred to an expression vector. Because of the nature of the genetic code, the cloned or hybrid gene or portions thereof will direct the production of the amino acid sequences for which it codes. The general techniques for constructing expression vectors with cloned genes located in the proper relationship to promoter regions are described by B. Polisky, et al., Proc. Natl. Acad. Sci. U.S.A. 73, 3900 (1976), K. Itakura, et al., Science 198, 1056-1063 (1977), L. Villa-Komaroff, et al., Proc. Natl. Acad. Sci. U.S.A. 75, 3727-3731 (1978) and others.
The term "expression" may be characterized in the following manner. Even in relatively simple prokaryotic organisms, the cell is capable of synthesizing many proteins. At any given time, many proteins which the cell is capable of synthesizing are not being synthesized. When a particular polypeptide, coded for by a given gene, is being synthesized by the cell, that gene is said to be expressed. In order to be expressed, the DNA sequence coding for that particular polypeptide must be properly located with respect to the control region of the gene. The function of the control region is to permit the expression of the gene under its control to be responsive to the changing needs of the cell at any given moment.
As used throughout this specification, the following definitions apply for purposes of the present invention:
A cloning vehicle is an extra-chromosomal length of duplex DNA comprising an intact replicon that can be replicated within a unicellular organism by transformation. Generally, cloning vehicles are derived from viruses and bacteria, and most commonly take the form of circular loops of bacterial DNA called plasmids.
The term gene refers to those DNA sequences which are responsible for the transmission and synthesis of a single protein chain.
The term infection refers to the invasion by pathogenic agents of cells where conditions are favorable for their replication and growth.
The term transfection refers to a technique for infecting cells with purified nucleic acids of viruses by precipitation of viral DNAs and uptake into cells upon addition of calcium chloride to solutions of DNA containing phosphate or other appropriate agents such as dextran sulfate.
A number of host-vector systems utilizing the above-described general scheme and techniques have been developed for use in the commercial or experimental synthesis of proteins by genetically modified organisms. Many of these host-vector systems are prokaryotic host-vector systems, such as that described in U.S. Pat. No. 4,338,397 to Gilbert, et al., and the system utilized for .beta.-interferon synthesis in Escherichia coli as described by T. Taniguchi, et al., Proc. Natl. Acad. Sci. U.S.A. 77, 5230 (1980) (see also D. V. Goeddel, et al., Nucleic Acid Res. 8, 4057 (1980) and Y. Mory, et al., Eur. J. Bio. Chem. 120, 197 (1981). Additionally, systems have been utilized which employ yeast as a vector such as the system employed for hepatitis B surface antigen synthesis as described by A. Miyanohara, et al., Proc. Natl. Acad. Sci. U.S.A. 80, 1 (1983), and the system for human interferon synthesis within yeast described by Pitzeman, et al., Science 219, 620 (1983).
The value of utilizing yeast or prokaryotic host-vector systems for the synthesis of desirable proteins is diminished by certain limitations inherent in such systems. For instance, the mRNA transcript or protein product of such systems may be unstable in the prokaryote. In addition, before a protein will be synthesized within a prokaryotic cell, the DNA sequence introduced into the microorganism must be free of intervening DNA sequences, nonsense sequences, and initial or terminal sequences which encode for polypeptide sequences which do not comprise the active eukaryotic protein. Further, some eukaryotic proteins require modification after synthesis (i.e., glycosylation) to become biologically active, and prokaryotic cells are generally incapable of such modifications.
Additional limitations associated with yeast or prokaryotic host-vector systems include the relatively low level of gene expression and the difficulties associated with the recovery of gene products synthesized from within the cell. U.S. Pat. No. 4,336,336 to Silhavy, et al., is specifically addressed to the problem of recovering the gene products, providing a method for synthesis and secretion of the protein by a genetically modified bacterium.
The use of viruses in eukaryotic host-vector systems has been the subject of a considerable amount of recent investigation and speculation. However, viral vector systems also suffer from significant disadvantages and limitations which diminish their utility. For example, a number of eukaryotic viral vectors are either tumorgenic or oncogenic in mammalian systems, creating the potential for serious health and safety problems associated with resultant gene products and accidental infection. Further, in some eukaryotic host-viral vector systems, the gene product itself exhibits antiviral activity, thereby decreasing the yield of that protein. Such was the case with the 80% reduction in the yield of simian virus 40 caused by only 100 units of interferon in the eukaryotic host-viral vector system described by D. Gheysen and W. Fiers, J. Molec. Applied Genet. 1, 385-394 (1982).
Another problem inherent in those eukaryotic host-viral vector systems currently utilized is presented by the fact that, because they have fewer restriction sites, it is easier to insert exogenous DNA into simple viruses at specific locations. However, because of the morphology of the virus, the amount of exogenous DNA which can be packaged into a simple virus is limited. This limit becomes a particularly acute problem due to the fact that eukaryotic genes, because they usually contain intervening sequences, are too large to fit into simple viruses. Further, because they have many restriction sites, it is more difficult to insert exogenous DNA into complex viruses at specific locations.
The present invention overcomes many of the limitations discussed above by utilizing a baculovirus and a promoter within the baculovirus genome to produce a viral expression vector in a eukaryotic host-vector system. More particularly, the baculovirus Autographa californica (AcMNPV) and the polyhedrin promoter may be utilized to produce a recombinant viral expression vector capable of extremely high levels of expression of a selected gene in a eukaryotic host insect cell. Additionally, the resultant gene products of this system may be efficiently secreted into the cell medium, alleviating those difficulties associated with the recovery of protein products. Further, and more significantly, this system is not oncogenic or tumorgenic in mammals. The theoretical advantages of utilizing baculoviruses in a eukaryotic host-viral vector system are discussed in more detail by L. K. Miller, "A Virus Vector for Genetic Engineering in Invertebrates." In: Panopoulos, N. J. (Ed.), Genetic Engineering in the Plant Sciences (New York, Praeger Publishers, 1981), pp. 203-224.